Aptamer-Modified Cu2+-Functionalized C-Dots: Versatile Means to Improve Nanozyme Activities-“Aptananozymes”

The covalent linkage of aptamer binding sites to nanoparticle nanozymes is introduced as a versatile method to improve the catalytic activity of nanozymes by concentrating the reaction substrates at the catalytic nanozyme core, thereby emulating the binding and catalytic active-site functions of native enzymes. The concept is exemplified with the synthesis of Cu2+ ion-functionalized carbon dots (C-dots), modified with the dopamine binding aptamer (DBA) or the tyrosinamide binding aptamer (TBA), for the catalyzed oxidation of dopamine to aminochrome by H2O2 or the oxygenation of l-tyrosinamide to the catechol product, which is subsequently oxidized to amidodopachrome, in the presence of H2O2/ascorbate mixture. Sets of structurally functionalized DBA-modified Cu2+ ion-functionalized C-dots or sets of structurally functionalized TBA-modified Cu2+ ion-functionalized C-dots are introduced as nanozymes of superior catalytic activities (aptananozymes) toward the oxidation of dopamine or the oxygenation of l-tyrosinamide, respectively. The aptananozymes reveal enhanced catalytic activities as compared to the separated catalyst and respective aptamer constituents. The catalytic functions of the aptananozymes are controlled by the structure of the aptamer units linked to the Cu2+ ion-functionalized C-dots. In addition, the aptananozyme shows chiroselective catalytic functions demonstrated by the chiroselective-catalyzed oxidation of l/d-DOPA to l/d-dopachrome. Binding studies of the substrates to the different aptananozymes and mechanistic studies associated with the catalytic transformations are discussed.

The ratios between the Cu 2+ -ions-functionalized C-dots and aptamer could be determined by the relative calibration curves. As we can see from the Figure S5A and B, the two calibration curves at 260 and 338 nm were formed by the different concentrations of the Cu 2+ -modified C-dots solution by UV spectra. Figure S5C and D show the UV spectra of 5-DBA and 5-TBA associated with Cu 2+ -ions-functionalized C-dots. After we attach the NH 2 -aptamers to the C-dots, the calibration curve at 330 nm is required to quantify the amount of C-dots, while the absorbance of C-dots at 260 nm is obtained. By the subtraction of the spectrum at 260 nm, the net absorbance of the nucleic acids associated with the C-dots is evaluated. It should be noted that multiple groups of experiments were executed to achieve the similar ratio of Cu 2+ -ions/aptamers to C-dots.

Kinetic measurements with aptananozymes
Kinetic measurements were performed at 25 °C using a Biotek Synergy H1 microplate reader equipped with a Biotek dual dispensing unit and using Corning 3696 96-well plates. For dopamine oxidation, the aptananozymes (0.2 μg mL −1 ) were dissolved in 5 mM MES buffer, pH 5.5, 5 mM MgCl 2 , 100 mM NaCl, and 10 μL of dopamine, consisting of variable concentrations which were added to the respective wells. Subsequently, 10 μL of H 2 O 2 (final concentration 1 mM) was dispensed into each well, and the absorbance values of the oxidized products (absorbance at 480 nm, ε = 3058 M −1 cm −1 ) were measured in the different wells for a time interval of 60 min. For the L-tyrosinamide oxidation process, the aptananozymes (0.5 μg mL −1 ) were dissolved S5 in in 50 mM phosphate buffer solution (pH 7.2, containing 100 mM NaCl and 5 mM MgCl 2 ), and 10 μL of L-tyrosinamide, consisting of variable concentrations which were added to the respective wells. Subsequently, 10 μL of H 2 O 2 (final concentration 5 mM) was dispensed into each well, and the absorbance values of oxidized products (absorbance at 475 nm, ε = 3600 M −1 cm −1 ) were measured in the different wells for a time interval of 120 min. As for the L-DOPA and D-DOPA oxidation process, the aptananozyme I (0.2 ug mL −1 ) was dissolved in in 50 mM phosphate buffer solution (pH 7.2, containing 100 mM NaCl and 5 mM MgCl 2 ), and 10 μL of L-tyrosinamide, consisting of variable concentrations which were added to the respective wells.
Subsequently, 10 μL of H 2 O 2 (final concentration 1 mM) was dispensed into each well, and the absorbance values of oxidized products (absorbance at 475 nm, ε = 3600 M −1 cm −1 ) were measured in the different wells for a time interval of 120 min.
In these experiments, the aptananozyme IV or the aptananozyme XIII was subjected as catalyst for the oxidation of dopamine or oxygenation of L-tyrosinamide, respectively. The catalysts run for four catalytic cycles, where the particles were separated by centrifugation after each catalytic cycle. Figure S8A shows the rates of oxidation of dopamine to aminochrome at variable concentrations of dopamine following four catalytic cycles. Figure S8B shows   S14 Figure S10 presents the raw heat plots (upper panels) and derived titration curves (lower panels) corresponding to (i) the scrambled DBA-aptananozyme and (ii)-(vi) the respective aptananozymes I-V.  Table S2. S15 Figure S11A presents the raw heat plots (upper panels) and derived titration curves (lower panels) corresponding to (i) L-DOPA-aptananozyme and (ii) D-DOPAaptananozyme.  Table S4.   S19 Figure S16 presents the raw heat plots (upper panels) and derived titration curves (lower panels) corresponding to (i) the scrambled TBA-aptananozyme and (ii)-(vi) the respective aptananozymes X-XIV.  Table S6.

S20
The chiral features of the tyrosinamide aptamer and its binding affinity towards L-     In this scheme, eq. (1) consistent with the experimental results demonstrating the formation of AA• by the catalyst, Figure 5A, panel II. The AA• induced formation of •OOH in the presence of H 2 O 2 , eq. (2), is supported by the formation of •OOH and AA•, Figure 5A, panel III. The resulting Cu I species are oxidized by H 2 O 2 to form the •OH that is rapidly depleted by the generation of AA•, eq. (3), and eq. (4), and the resulting AA • is feedback into reaction (2) that generates the peroxy • OOH oxygenation species. Figure S21. High-resolution XPS spectra of Cu (A, Cu 2+ -ions-functionalized C-dots before reacting with AA  and H 2 O 2 ; B, Cu 2+ -ions-functionalized C-dots after reacting with AA  and H 2 O 2 ).